The long-range goal of this research program is to elucidate the mechanisms by which the polypeptide hormone epidermal growth factor (EGF) stimulates cell replication. As a first step toward this goal, we have studied the fate of cell-bound EGF in cultured cells. EGF binds to its receptor on the cell surface, clusters in coated pits, is internalized in endocytic vesicles and eventually is degraded in lysosomes. Experiments outlined in this project will use biochemical and morphological methods to investigate two questions raised by the above results. (1) What role does internalization play in the biological action of the hormone? (2) What are the biochemical and cellular mechanisms involved in clustering and internalizing EGF? Preliminary indirect evidence suggests that a phospholipase A2-like activity is involved in EGF internalization. We will directly test the hypothesis that this enzyme is involved in either the clustering or endocytosis of EGF. In a separate set of experiments, we will determine if EGF receptors that are clustered in coated pits are crosslinked either to other EGF receptors or to adjacent membrane components. The clustering of the EGF receptors also will be studied in subcellular vesicles in order to determine the minimum number of cellular structures required for clustering to occur. Recently, it has been shown that human carcinoma cells A-431 contain a membrane-bound tyrosinespecific protein kinase that is stimulated by EGF and closely associated with the EGF receptor. These results raise the third question to be studied. What is the in situ molecular weight of: (1)\the functional component responsible for EGF binding; and (2)\the tyrosine-specific protein kinase that is associated with the EGF receptor? This will be investigated using high energy radiation inactivation. The goal is to determine if the EGF receptor and the protein kinase are the same protein or are different proteins that are closely associated. This is of particular interest in light of recent evidence that the EGF receptor is antigenetically related to the transforming protein of Rous sarcoma virus.